Combined Haploinsufficiency for ATM and RAD9 As a Factor in Cell Transformation, Apoptosis, and DNA Lesion Repair Dynamics

Research Article

Combined Haploinsufficiency for ATM and RAD9 as a Factor in Cell Transformation, Apoptosis, and DNA Lesion Repair Dynamics

Lubomir B. Smilenov, Howard B. Lieberman, Stephen A. Mitchell, Ronald A. Baker, Kevin M. Hopkins, and Eric J. Hall

Center for Radiological Research, College of Physicians and Surgeons, Columbia University, New York, New York

Abstract These data as well as the fact that most mice heterozygous for DNA Loss of function of oncogenes, tumor suppressor genes and repair genes have the same life span as the wild-types when not DNA damage processing genes has been implicated in the challenged with mutagens, strongly suggest that haploinsufficiency development of many types of cancer, but for the vast majority is a critical factor in the cellular response to stress conditions, and of cases, there is no link to specific germ line mutations. In the even more importantly, individuals with different genotypes last several years, heterozygosity leading to haploinsufficiency respond differently to the same environmental challenges. for proteins involved in DNA repair pathways was shown to In the case of heterozygosity when one allele of a gene is play a role in genomic instability and carcinogenesis after inactivated, predisposition to transformation is based on a more DNA damage is induced. Because the effect of haploinsuffi- probable frequency of mutations than on the complete inactivation ciency for one protein is relatively small, we hypothesize that of both alleles of cancer-related genes such as RB1, p53, BRCA1,or predisposition to cancer could be a result of the additive effect BRCA2. Epidemiologic studies indicate that only 15% to 20% of of heterozygosity for two or more genes, critical for pathways familial breast cancer cases, for example, are a result of mutation that control DNA damage signaling, repair or apoptosis. To in BRCA1 or BRCA2 (12, 13). The rest are most probably due to address this issue, primary mouse cells, haploinsufficient for genetic factors unlikely to involve a mutation in a highly related one or two proteins, ATM and RAD9, related to the cellular tumorigenesis gene (14). Evidence suggests that the risk might be response to DNA damage were examined. The results show based on the additive contribution of several factors, each that cells having low levels of both ATM and RAD9 proteins individually having a small effect (15–17) difficult to determine are more sensitive to transformation by radiation, have when present alone. different DNA double-strand break repair dynamics and are We hypothesize that predisposition to cancer could be a result less apoptotic when compared with wild-type controls or of the additive effect of heterozygosity for two or more genes, those cells haploinsufficient for only one of these proteins. critical for pathways that control DNA damage signaling, repair, Our conclusions are that under stress conditions, the or apoptosis. Because in many cases heterozygosity leads to efficiency and capacity for DNA repair mediated by the haploinsufficiency (18), we suggest that the function of signaling ATM/RAD9 cell signaling network depend on the abundance networks that impact on maintaining genomic integrity depends of both proteins and that, in general, DNA repair network on the proper amounts of key proteins and that haploinsufficiency efficiencies are genotype-dependent and can vary within a can lead to conditions where network efficiency under stress is specific range. (Cancer Res 2005; 65(3): 933-8) suboptimal. This might result in a decrease in the effectiveness of processes related to suppression of tumor initiation, such as Introduction apoptosis and the efficient processing of DNA damage. To address this issue, cells haploinsufficient for one or two In the last few years, mounting evidence suggests that proteins related to the cellular response to DNA damage—ATM heterozygosity leading to haploinsufficiency for proteins involved and RAD9—were examined. Both proteins are important factors in in DNA repair pathways plays a role in genomic instability and DNA double-strand break repair (19, 20), they rapidly colocalize to carcinogenesis. Haploinsufficiency for p53, PTEN, BubR1, NBS1, regions containing DNA double-strand breaks after DNA damage H2AX, p18(INK4c), BLM, Rb, APS, and ATM has been shown to be (21, 22), and ATM can phosphorylate RAD9 (23). We generated an important factor in carcinogen-induced tumors (1–11). Most mice haploinsufficient for each or both proteins, and analyzed of these genes code for tumor suppressor proteins. A major isolated mouse embryo fibroblasts (MEFs) and thymocytes to conclusion from these data is that, contrary to one of the current monitor three end points related to tumorigenesis-cell transfor- views on tumorigenesis, inactivation of one allele of a tumor mation, apoptosis and DNA double-strand break repair. The results suppressor gene is enough to contribute to tumor progression. show that cells having lower amounts of both ATM and MRAD9 Another conclusion from most of the cases is that animals or cells are more sensitive to transformation induced by radiation, have haploinsufficient for the specified proteins have higher transfor- different dynamics of DNA double-strand break repair, retain more mation rates after DNA damage is induced, but when their DNA is double-strand breaks after radiation exposure and are less not significantly damaged by exogenous sources, tumor develop- apoptotic than the wild-type control, or cells haploinsufficient for ment rates are the same as for their wild-type counterparts. only one of these proteins. Our conclusions are that, under stress conditions, the efficiency and capacity for DNA repair mediated by the ATM/RAD9 cell-signaling network depends on the expression Requests for reprints: Lubomir B. Smilenov, Center for Radiological Research, levels of both proteins and that, in general, DNA repair network College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032. Phone: 212-305-5661; Fax: 212-305-3229; E-mail: [email protected]. efficiencies are genotype-dependent and can vary within a specific I2005 American Association for Cancer Research. range. These findings point to the possibility of estimating an www.aacrjournals.org 933 Cancer Res 2005; 65: (3). February 1, 2005

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Cancer Research individual’s susceptibility to health risks associated with carcino- (or 24 hours where indicated), the proportion of apoptotic cells was gen exposure based on genotype and levels of specific proteins. measured by staining with Annexin V-PE and 7-AAD according to the manufacturer’s instructions (PharMingen, San Diego, CA). DNA Lesion Visualization. MEFs were isolated, cultured close to the Materials and Methods stage of senescence, and then plated in two-well slide chambers (Lab-Tek, 4 Mice. Atm and Rad9 wild-type (wt), heterozygous (hz), and knockout Naperville, IL) at a density of 3 Â 10 /well. After 2 days, the cells were (ko) mice used in the experiments were described previously (24, 25). Both irradiated with 0.5 Gy of g-rays, fixed for 10 minutes in 2% paraformalde- mouse mutant heterozygous genotypes caused haploinsufficiency for the hyde, permeabilized for 20 minutes in methanol at À20jC, blocked for 1 corresponding proteins (11, 25). The two groups of mice were mated and hour in 5% goat serum, and stained with rabbit anti-g-H2AX antibody (a gift only F1 littermates were used. Genotypes were determined by PCR (24, 25). from Dr. W. Bonner) for 2 hours. The bound antibody was visualized using Mice heterozygous for Atm or Rad9, or the double heterozygous animals, Alexa Fluor goat anti-rabbit antibody (Molecular Probes, Eugene, OR), and displayed no detectable abnormalities through 12 months of age. cell nuclei were stained with PI/RNase solution (PharMingen). Slides were Embryo Cell Preparation. Pregnant females were sacrificed on day 14 viewed on a laser scanning confocal microscope (Nikon Co., Tokyo, Japan). of gestation. The embryos were surgically removed and embryonic tissue At least 100 cells were scored, and the average number of foci per cell was prepared in culture. Each embryo was cultured separately, and during the 4 calculated. days necessary to amplify MEF cells in mass culture, they were genotyped. Cell Transformation Assay. Exponentially growing MEFs received a Results dose of 2 Gy of g-rays in an acute exposure, and controls were sham- irradiated. MEFs were then plated in 100-mm plates at a density of 6,000 Cell Transformation Assay. Radiation-induced transformation cells/plate over a feeder layer of 70,000 cells prepared from the same of MEFs was examined to begin to access the impact of genotype embryo but irradiated previously with a supralethal dose. After 2 weeks of on this end point. A total of 21 embryos from five litters were used growth in DMEM medium supplemented with 10% fetal bovine serum at and included five for genotypes Atmwt/Mrad9wt, Atmhz/Mrad9wt, 37jC in a 5% CO air-humidified incubator, cells were fixed, stained, and 2 and Atmwt/Mrad9hz and six for Atmhz/Mrad9hz. Yields of yields of transformed clones scored. The scoring criteria were developed and examined by preliminary experiments, where embryo cells were transformed clones were measured both for unexposed controls irradiated and plated with the same density. The clones which seemed and after a dose of 2 Gy. The results shown in Tables 1 and 2 dense and had stellate-shaped piled cells were isolated with cloning indicate a statistically significant higher transformation frequency cylinders. These clones were expanded and injected into nude mice. Those for the double heterozygous cells. Transformation frequencies for that caused the development of tumors were designated as transformed. these cells are more than double that of the wild-type population. Clones that matched their shape and dimensions were scored as The Mrad9 heterozygous cells show a transformation frequency transformed in later experiments. Plating efficiency, cell surviving fractions, close to that of the wild-type cells, and the frequency for the ATM and the spontaneous and radiation-induced frequency of morphologic heterozygous cells is between the wild-type and double heterozy- transformation were determined. Clonogenic Survival. To assess clonogenic survival, MEFs were exposed gous cells. There were small differences in the clonogenic survival to different doses of g-rays. Following irradiation, sufficient numbers of cells for all populations after irradiation (Fig. 1). were plated into 100 mm culture dishes so that accounting for plating Apoptosis of Thymocytes. We examined thymocytes from efficiency and surviving fraction following radiation, f100 viable cells single and double heterozygous animals for radiation-induced would be present in each dish. The total number of cells (viable plus feeder) apoptosis. The number of animals and the genotypes used were, was 70,000 per dish. Dishes were incubated for 2 weeks without a medium respectively: Atmwt/Rad9wt (five), Atmhz/Rad9hz (seven), Atmwt/ change, and the resulting colonies were stained with Giemsa to determine Radhz (five), Atmhz/Rad9wt (six), Atmko/Rad9wt (three). The both the plating efficiencies and surviving fractions of control and mice were from four different litters. The results show differences irradiated cells. Data from a minimum of three independent experiments in apoptotic frequencies related to genotype (Fig. 2). Wild-type cells were pooled. All data for clonogenic survival were presented as a mean display the highest apoptotic frequencies after irradiation, whereas together with SE. Apoptosis and Cell Survival Assays. Wild-type and heterozygous mice, ATM-deficient cells show the lowest. The differences between at the age of 2 months, were sacrificed, and thymocytes were isolated after apoptotic frequencies in Atm wild-type and heterozygous cells were careful thymus homogenization. Thymocytes were seeded into 24-well small but statistically significant and show that Atm heterozygosity plates (in RPMI/10% fetal bovine serum) at a density of 5 Â 106/mL could be a factor influencing programmed cell death. Mrad9 and exposed to g-radiation at doses of 1, 2, 4, and 8 Gy. Six hours later heterozygous cells show the same apoptotic rates as the wild-type

Table 1. Transformation frequencies of unirradiated or irradiated cells differing in the status of Atm and Mrad9

Genotype Dose (Gy) Total number Number of Transformed of clones scored transformed clones clones (%)

Atmwt/Mrad9wt 0 31,240 5 0.02 2 22,800 24 0.11 Atmwt/Mrad9hz 0 28,450 5 0.02 2 16,470 27 0.16 Atmhz/Mrad9wt 0 35,170 5 0.01 2 16,720 35 0.21 Atmhz/Mrad9hz 0 27,830 13 0.05 2 18,900 63 0.33

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Table 2. Comparisons of radiation-induced transformation knockout cells had the highest background staining and showed between MEFs of different genotypes versus wild-type slow foci formation, and high residual foci frequencies after 24 hours. MEFs Discussion Atmhz/ Atmwt/ Atmhz/ We showed that cells haploinsufficient for both Atm and Mrad9 Mrad9wt Mrad9hz Mrad9hz are more sensitive to transformation induced by radiation, show different dynamics of double-strand break repair, retain more Relative transformation 1.91 1.45 3.10 double-strand breaks after radiation exposure, and are less (2 Gy) apoptotic than wild-type cells or those haploinsufficient for only t test (2 Gy) P = 0.03 P = 0.31 P = 0.0001 one of the encoded proteins. Haploinsufficiency as a result of heterozygosity for tumor suppressor genes in combination with NOTE: Relative transformation is defined as the ratio of the number of carcinogens has been implicated in tumorigenesis (5–12). Our transformed clones per surviving heterozygous cells relative to the number of transformed clones per surviving wild-type cells. The previous results show that heterozygosity for Atm modestly statistical significance of differences in transformation frequency increased the transformation of MEFs after irradiation (11), and between the various cells with heterozygous genotypes and wild-type was also a factor in cataract formation (26). Heterozygosity for Atm cells was analyzed by Student’s t test. has long been suspected as a contributing factor in familial breast cancer as well as other types of cancer (12). Theoretically, all familial cases of tumor development where one protein is haploinsufficient or deficient, points to other important factors control. Remarkably, the apoptotic frequencies were significantly characteristic for the family. Because usually no mutations in other reduced in the double heterozygous cells. The results for this genes have been identified in many of these cases, we hypothesize genotype are closer to those obtained for the Atm null cells than for that some of these unknown factors could be a second the wild-type, showing that haploinsufficiency for two functionally haploinsufficiency for a protein related functionally to the first related proteins may have an additive negative effect on pathways one. In this respect, we analyzed three cancer-related events: cell where both proteins are normally involved. transformation, apoptosis, and double-strand break repair in cells We measured apoptosis in thymocytes 6 hours after irradiation single- or double-haploinsufficient for Atm and Mrad9. We found to avoid high background apoptotic frequencies that are in the that haploinsufficiency for both proteins had an additive effect, range of 25% to 30% for these cells, 24 hours after isolation. A noticeably increasing cell transformation after radiation-induced concern was that results at 6 hours could represent only delayed DNA damage, and decreasing apoptotic frequencies in irradiated apoptosis for the double heterozygous thymocytes relative to the thymocytes, bringing the apoptotic levels closer to that observed in wild-type because activation of ATM-dependent pathways is the Atm null phenotype. Additionally, the double-haploinsufficient considered the first response to irradiation followed by activation phenotype changed the dynamics of double-strand break repair of other compensatory DNA damage repair pathways. To address and decreased the efficiency of removal of those lesions, as shown this, survival and apoptotic frequencies for Atmwt/Mrad9wt and by staining for g-H2AX. These data are consistent with a model Atmhz/Mrad9hz thymocytes were also measured 24 hours after indicating that low levels of ATM and MRAD9 proteins result in a irradiation. The results confirm the higher survival for double relatively inefficient cellular response to excessive DNA damage, heterozygous cells in comparison to the wild-type counterparts, indicating that differences in apoptosis are maintained for longer periods of time (Fig. 3). Because ATM is an important factor in T cell differentiation, thymocytes from all animals were examined for CD4/CD8 markers. No difference was found in double positive and single positive distributions except for the ATM-deficient thymocytes where, as expected, a partial block at the CD4/CD8 double positive stage and reduced numbers of single positive CD4 and CD8 cells were found (data not shown). DNA Double-Strand Break Repair Dynamics. The appearance of DNA double-strand breaks and their repair in MEFs having different genotypes were examined. Cells were passaged until close to senescence because fast proliferating early passage MEFs show high background g-H2AX staining. Changes in the number of foci formed in response to 0.5 Gy of g-rays were followed for up to 24 hours (Fig. 4). The genotypes evaluated were Atmwt/ Mrad9wt, Atmwt/Mrad9hz, Atmhz/Mrad9wt, Atmhz/Mrad9hz, and Atmko/Mrad9wt. There were no significant differences betweenthenumberoffociinthewild-typeandsingle heterozygous cells. g-H2AX foci formation was slower in the Atmhz/Mrad9hz cells but after 2 hours were statistically equal to those shown by cells with the other genotypes. After 24 hours, Figure 1. Clonogenic survival of MEF cells irradiated with g-rays. Points, means the Atmhz/Mrad9hz cells show more residual double-strand from at least three independent experiments; bars, SE. Surviving fractions breaks than wild-type and the single heterozygous cells. The Atm measured at the doses tested were fitted with the linear-quadratic equation. www.aacrjournals.org 935 Cancer Res 2005; 65: (3). February 1, 2005

significant accumulation of mutations and retention of damage, which are the reasons for subsequent accelerated cell death by mitotic catastrophe. Depending on the magnitude of DNA damage, in some cases, a small fraction of cells are able to escape, continue to divide and can develop into tumors. This scenario is well supported by the inevitable development of thymic lymphomas in all Atm knockout mice. Although they develop in unirradiated as well as irradiated mice, the lack of proper end joining for V(D)J recombination is similar to the lack of DNA double-strand break repair generated by radiation, and illustrates the link between the accumulation of mutations, improper apoptosis, and tumor development. Our results show that double heterozygous thymocytes were much less apoptotic than wild-type and single heterozygous cells. Applying the same logic as to the Atm knockout thymocytes, we suggest that the rate of mutation accumulation in these cells should be higher than in the wild- Figure 2. Apoptosis of thymocytes having different genetic backgrounds. type and single heterozygous cells, and their respective oncogenic Thymocytes from mice were irradiated with different doses of g-rays and transformation potentials should also be higher. These results apoptosis was measured 6 hours after irradiation. The percentage of apoptotic cells at 0 Gy were subtracted from the rest of the data points for each genotype. could explain the higher transformation frequency we observe in the double-haploinsufficient MEFs. Indeed, transformation and apoptotic death seem to be inversely related. including DNA repair and apoptosis. The mechanisms behind these In another approach, we estimated the dynamics of double-strand events could be related to specific functions of ATM and MRAD9 in break repair in MEFs by visualizing the phosphorylated form of DNA repair and signal transduction networks. ATM is a sensor/ histone g-H2AX. As was shown previously, g-H2AX is an important transducer protein involved in the initial response to DNA double- marker for detection of double-strand breaks (34) and was used for strand breaks. The protein is activated immediately after DNA evaluating DNA repair dynamics in different types of cells including double-strand break induction (27) and is subsequently involved in MEFs (35). Our results demonstrate that MEFs haploinsufficient for a large group of events, including downstream signaling by ATM and RAD9 show different repair dynamics than wild-type cells activating DNA repair, checkpoint, and apoptotic control proteins. and accumulate a higher number of residual double-strand breaks Atm knockout organisms develop progressive cerebellar ataxia, after irradiation. This could indicate higher frequencies of mutation lymphoma and leukemia. They are characterized by chromosomal accumulation for cells with that specific genotype. The mechanisms instability and hypersensitivity to ionizing radiation (19, 28). The underlying these results could be related to less efficient DNA role of RAD9 is less well established but we know that it forms a damage detection, repair, and apoptosis. heterotrimer with RAD1 and HUS1 (29, 30), a complex believed to All of these results indicate that haploinsufficiency for two be critical for cell cycle checkpoint control. Both ATM and the closely interacting proteins (i.e., ATM and MRAD9) functioning in RAD9 complex colocalize at points of double-strand breaks related pathways could play a significant role in tumorigenesis by minutes after DNA damage is incurred, as part of a large protein altering the mechanisms preventing it. Another conclusion is that complex involving TopBP1, RAD50, RAD9, ATM, and BRCA1 (21, the effectiveness of DNA repair networks may vary within a specific 22), and ATM can phosphorylate RAD9 (23). These data indicate range depending on the genotype and levels of the proteins strong cooperation between RAD9 and ATM proteins during the initial events of the cellular response to DNA damage. Our previous results (11) show increased sensitivity to radiation oncogenesis in MEFs haploinsufficient for the ATM protein. We used the same approach estimating the transformation frequency of MEFs haploinsufficient for both ATM and RAD9 proteins. Surprisingly, the double-haploinsufficient cells were significantly more sensitive to radiation oncogenesis than were the corresponding wild-type cells by a factor of about 3. The transformation frequency of the double heterozygous cells were also higher than for the cells haploinsufficient for only ATM or RAD9 proteins. Comparison of the RAD9-haploinsufficient MEFs and wild-type MEFs didn’t show statistically significant differences. ATM-haploinsufficient cells were more sensitive in comparison with the wild-type MEFs by a factor of 2. Clonogenic survival results were similar in the range from 1 to 6 Gy for all genotypes tested. The function of ATM in triggering apoptosis is well established. Our data shown here as well as that of others (31–33) indicate that thymocytes deficient for ATM are less apoptotic after irradiation in Figure 3. Thymocyte survival 24 hours after irradiation. Thymocytes were comparison to wild-type cells. This is a result of the lack of isolated from mice, irradiated, and incubated overnight at a density of 5 Â 106 cells/mL. Twenty-four hours later, 100 AL of the cell suspension was labeled with detection of double-strand breaks by ATM, and lack of activation anexin V/7-AAD. Each sample was measured by flow cytometry for 30 seconds. of cell cycle checkpoints and apoptosis. The consequences are a Cells not stained by the annexin-V or 7AAD are shown as live nonapoptotic cells.

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Currently, there are two basic paradigms for the initial stage of tumorigenesis-mutator phenotype and aneuploidy (36, 37). According to the mutator phenotype hypothesis, the initial stages of carcinogenesis are a result of mutation of genetic stability genes which increase mutation rates for other genes, and eventually lead to cell transformation. The aneuploidy model suggests that mutation of a small number of genes required for cell division leads to chromosome breaks or unequal chromosome segregation. This results in genetic instability and the generation of mutations in multiple genes. The aneuploidy model explains well how small numbers of initial mutations could lead to the high subsequent number of mutations needed for cancer progression. A problem with both models is the inability to explain how the initial mutations occur because efficiency of DNA repair networks in normal cells is very high and the probability of complete inactivation of even three to five relevant genes in the course of the lifetime of a cell is very low (38). Our results suggest Figure 4. Double-strand break repair in different genetic backgrounds. MEFs having the indicated genotypes were irradiated with 0.5 Gy of g-rays. The that DNA repair pathway effectiveness could be significantly number of DNA double-strand breaks were revealed by staining with anti g-H2AX lowered by a few mutations affecting one copy of related genes. antibody. Columns, average of at least 100 cells counted for each genotype at This, especially when combined with the presence of environ- the specific time points indicated. Bars, SE from two independent experiments. The Atmhz/Mrad9wt and Atmwt/Mrad9hz MEFs showed results similar to mental mutagens, may result in a high number of DNA lesions, the Atmwt/Mrad9wt number of foci (results not shown). mutation accumulation and tumor initiation. This model could support the conclusion that each individual has unique inherent sensitivity to mutagens, depending on the levels or activity of the involved. An additive effect of a second haploinsufficiency may proteins involved in DNA repair or related processes. This significantly contribute to the destabilization of a particular cell sensitivity could be measured in vitro by the analysis of genotype signaling network. Therefore, it seems that cell signaling networks and levels of specific proteins. This could lead to the are finely tuned and are most effective only when the concentration establishment of individual limits for mutagen exposure that will and consequently the activity of the proteins involved are at bear a health risk, and may thus have significant consequences optimal levels. As a result, the capacity for DNA repair under stress for cancer prevention. conditions will depend on an individual’s genotype, and the degree of accumulation of mutations in the presence of the same dose of mutagens could vary from individual to individual. Acknowledgments Interpretation of these findings in the context of tumor Received 6/1/2004; revised 11/22/2004; accepted 11/30/2004. initiation and progression leads to the suggestion that initial Grant support: RSNA Seed Grant Program (to L. Smilenov), Avon Scholar Pilot eventsintransformationcouldariseinaheterozygous Awards in Breast Cancer (to L. Smilenov), NASA grant NAG 9-1519 (to E.J. Hall), U.S. DOE grant No. DE-FG02-03ER63629 (for E.J. Hall and S.A. Mitchell) and NIH grants background of partially destabilized networks and the presence CA89816 and GM52493 (6M52493) (to H.B. Lieberman). of mutagens. Mutations where one copy of a gene is inactivated The costs of publication of this article were defrayed in part by the payment of page are much more probable than complete gene inactivation. charges. This article must therefore be hereby marked advertisement in accordance Therefore, the additive effect of two or more haploinsufficiencies with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Drs. Olga Sedelnikova and W. Bonner, National Cancer Institute, NIH, for may be a decisive factor in the initial accumulation of damage providing us with the g-H2AX antibody and for the methodological help regarding leading to cancer. these experiments.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Combined Haploinsufficiency for ATM and RAD9 as a Factor in Cell Transformation, Apoptosis, and DNA Lesion Repair Dynamics

Lubomir B. Smilenov, Howard B. Lieberman, Stephen A. Mitchell, et al.

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